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Abstract

Femtosecond laser at 780 nm excitation wavelength was used to photo-convert the physiological sarcomeric single band (SB) second harmonic generation (SHG) pattern into double band (DB) in Xenopus laevis premetamorphic tail muscles. This photo-conversion was found to be a third order non-linear optical process and was drastically reduced at 940 nm excitation wavelength. This effect was no longer observed in paraformaldehyde fixed muscles and was enhanced by hydrogen peroxide. The action of hydrogen peroxide suggests that reactive oxygen species (ROS) could contribute to this photo-conversion. These results demonstrate that sarcomeric DB SHG pattern is a marker of sarcomere photodamage in xenopus tadpole muscles and highlight the need of being very careful at using two-photon excitation while observing living tissues. Moreover they open new avenues for in situ intravital investigation of oxidative stress effects in muscle dysfunctions and diseases.

Figures (5)

Fig. 1 Typical SHG images illustrating the laser-induced SB to DB sarcomeric SHG pattern conversion obtained at 780 nm excitation wavelength. (a) and (b) are examples of pre- and post-conversion single frame protocol images obtained respectively before and after the photo-conversion time-lapse protocol (c) (see Experimental Methods section). In (c), thumbnail strips are representative of the photo-conversion time-lapse sequence (left- right and top-down) and were realized in the dot-delimited area in (a) and (b). (d) Raw pixels intensity (Ipix) profiles (8-bit scale) along indicated lines in the first and the last frames in (c). Note that the first and last thumbnail and their corresponding intensity profiles at indicated lines are labeled “0 s” and “286 s” which correspond to their acquisition time within the sequence. Note the progressive spatiotemporal (left to right) “wave-like” propagation of sarcomere SHG pattern duplication within the sequence. Both thumbnail and intensity profile plots indicate a great contrast reduction suggestive of photodamage. This photodamage is accompanied by a SB to DB conversion which was slightly propagated beyond the zoom area in (b) in an anisotropic manner, following the long axis of myofibrils.

Fig. 3 Influence of the laser mean power intensity (I) on SB to DB sarcomeric SHG pattern photo-conversion kinetic. The incident laser power was varied and the half-time (τ1/2) until 50% SB to DB conversion was determined. Each symbol represents value from distinct field of view. Care was taken to maintain the focal plane at the same z position within the sample. The logarithm of the half-time latency τ1/2 was plotted against the logarithm of the laser power intensity (I) at the output of the objective lens. Fits were obtained by linear regression with the following equation: log (τ1/2) = 5.8 – 3.1log (I); R = 0.94.

Fig. 4 Laser-induced alteration of sarcomeric SHG pattern at 940 nm excitation in the presence of hydrogen peroxide (5 µL, 90 mM) injected in the heart of tadpoles. (a) and (b) are examples of pre- and post-conversion single frame protocol images obtained respectively before and after the photo-conversion time lapse protocol (c) (see Experimental Methods section). (c) Thumbnail strips representative of the photo-conversion time lapse sequence (from left to right) realized in the dot-delimited area in (a) and (b).(d) Raw pixels intensity (Ipix) profiles (8-bit scale) along indicated lines in the first and the last frames. Note that the first and last thumbnail and their corresponding intensity profiles are labeled “0 s” and “520 s” which correspond to their acquisition time within the sequence.

Fig. 5 Laser-induced alteration of sarcomeric SHG pattern at 780 nm excitation in the presence of hydrogen peroxide (5 µL, 90 mM) injected in the heart of tadpoles. (a) and (b) are examples of pre- and post-conversion single frame protocol images obtained respectively before and after the photo-conversion time lapse protocol (c) (see Experimental Methods section). (c) Thumbnail strips representative of the photo-conversion time lapse sequence (from left to right) realized in the dot-delimited area in (a) and (b). (d) Raw pixels intensity (Ipix) profiles (8-bit scale) along indicated lines in the first and the last frames are presented below the strips. Note that the first and last thumbnail and their corresponding intensity profiles are labeled “0 s” and “52 s” which correspond to their acquisition time within the sequence. Note the dark central zone of (b) corresponding to the dot-delimited area scanned during the photo-conversion time lapse protocol. The double headed continuous and dotted arrows represent respectively the favorable and unfavorable propagation direction of the effect of the laser outside the scanned area. Note that the propagation direction is quite parallel to the myofibril axis.